cDNA encoding a gene BOG (B5T over-expressed gene) and its protein product

ABSTRACT

Nucleic acids that encode novel polypeptides, designated in the present application as “BOG” (B5T Over-expressed Gene) are provided. BOG binds to pRb and is over-expressed in a number transformed rat liver epithelial (RLE) cell lines resistant to the growth inhibitory effect of TGF-β1 as well as in primary liver tumors. Compositions including BOG chimeras, nucleic acids encoding BOG and antibodies to BOG are also provided. Methods of using BOG to modulate pRb-protein interactions and to alter cellular phenotype are further provided.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/637,746,filed Aug. 11, 2000, now U.S. Pat. No. 6,727,079 issued Apr. 27, 2004which is a continuation of international application No. PCT/US99/04142,filed on Feb. 25, 1999, which claims priority to U.S. Ser. No.60/075,922, filed Feb. 25, 1998 and to U.S. Ser. No. 60/079,567, filedMar. 27, 1998, which applications are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTION MADE UNDER FEDERALLY SPONSORED RESEARCHAND DEVELOPMENT

The work performed during the development of this application utilizedsupport from the National Institutes of Health. The United Statesgovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the isolation andcharacterization of novel DNA and polypeptides, designated herein as“BOG”.

BACKGROUND OF THE INVENTION

Many types of human cancer are now believed to be caused by an imbalanceof growth regulators within a cell. A decrease in negative controlgrowth regulators and/or their deactivation can cause a cancerouscondition. Further, an increase in positive control growth regulatorscan also cause a cancerous condition.

Since the identification of the first tumor suppressor gene, much effortin cancer research has been focused on the identification of cellularproteins which interact with tumor suppressor proteins and theirinvolvement in human cancer. Moreover, many types of human cancers arethought to develop via mechanisms which impair the function of tumorsuppressor genes.

One of the most studied tumor suppressor genes is the retinoblastomasusceptibility gene (RB), whose gene product (pRb) has been shown toplay a key role in the regulation of cell division. R. A. Weinberg,Science, 254:1138 (1991). pRb is a nuclear protein that acts as a cellcycle control checkpoint by inhibiting G1/S progression. In interphasiccells, pRb contributes to maintaining the quiescent state of the cell byrepressing transcription of genes required for the cell cycle throughinteraction with transcription factors, such as E2F (see e.g. Hiebert etal., Genes Develop., 6, 177-185 (1992)). Upon entrance into the cellcycle, pRb is phosphorylated by cell cycle-dependent kinases (see e.g.Matsushime et al., Nature, 35, 295-300 (1992)) which is thought topermit its dissociation from transcription factors and, hence, theexpression of genes required for progression through the cell cycle. Theassociation of pRb with cell cycle regulators like cyclins and cellcycle-dependent kinases suggests a universal character to its function.

Deletion or inactivation of both RB alleles is an essential,rate-limiting step in the formation of retinoblastoma and osteosarcomathat arise within families that carry a mutated RB gene. RB inactivationis also found in other sarcomas, small cell carcinoma of the lung, andin carcinoma of the breast, prostate and bladder. The restriction ofpRb's involvement in human cancer to a limited number of tumor typessuggests that its hypothetical universal function is influenced by othergene products in a cell type-specific manner. Consistently, knock out ofthe RB gene in mice affects only specific cell types after several daysof embryonic development (see e.g. Clarke et al., Nature, 359, 328-330(1992)). The loss of this activity can induce cell transformation asevidenced by the reversion of the transformed phenotype in pRb cellsafter replacement of a functional pRb. Moreover, injection of the RBgene product, pRb, into G1 cells can block cell cycle progression. Thusthe identification of factors that interfere with and/or control pRbfunction is critical for understanding both cell cycle control andoncogenesis.

A major advance in the search for pRb function was the finding that pRbis a target for oncogenic products of DNA tumor viruses. Initial studiesdemonstrated that the adenovirus E 1A protein forms a complex with Rbwhich is dependent on sequences in the E1A protein. P. Whyte, et al.,Nature, 334:124 (1988). Certain transforming proteins such as SV40T andE7, which are derived from the transforming or tumor-associated subtypesof adenoviruses and human papilloma viruses (HPV) are also capable ofbinding to pRb, thereby blocking its normal function. All these viraloncoproteins interact with pRb through an LXCXE motif (where X is anyamino acid). Interaction of these viral proteins with pRb appears to bean important aspect of their oncogenic potential in which aninactivation of pRb function is achieved, that is equivalent to adeletion or mutation of RB. Growth factors such as transforming growthfactor-β1 (TGF-β1) also exert their cell cycle control via pRb. A. B.Roberts, et al., Peptide Growth Factors And Their Receptors, 421-427(1990). The mechanism(s) by which TGF-β1 inhibits cellular proliferationinvolves an attenuation of phosphorylation of pRb at the G1/S transitionof the cell cycle. See e.g. I. Reynisdottir, et al. Genes Dev., 9:1831(1995).

The ability of several transforming proteins from human DNA tumorviruses to activate cell proliferation has been a useful tool for theidentification of cellular factors involved in the regulation of thecell cycle. Negative regulators of cell growth may thus be effectivetargets for inactivation by these viral proteins, as it occurs with theproduct of the retinoblastoma gene. Adenovirus E1A, SV40 T antigen, andpapillomarivus E7 are three exemplary viral proteins which have beenfound to bind to pRb. This binding is responsible for the release oftranscription factors required for the expression of cell cycle genes(see e.g. Nevins, Science. 258, 424-429 (1992).

A conserved motif found in the three viral proteins allows forinteraction and complex formation with pRb (Moran, Curr. Op. Gen. Dev.,3, 63-70 (1993)). In the case of the adenovirus E1A protein, this motifis located in the transforming domain 2, which is required for growthactivation. The pRb-related product p107 also binds in this region.Domain 2 is also the site of interaction of an additional E1A-bindingprotein, p130. This has led to the suggestion that p130 has a structuralrelationship to pRb and p107 (Moran, Curr. Op. Gen. Dev., 3, 63-70(1993)).

The viral oncoprotein-binding domain in pRb, p107 and p130 is aconserved region termed the “pocket region” (see e.g. Ewin et al., Cell,66, 1155-1164 (1991)), and it is thought to play a primary role in thefunction of these proteins. The pocket is structurally formed by tworegions A and B, which are conserved in pRb, p107 and p130 and separatedby nonconserved spacers of different sizes in pRb, p107 and p130.

In addition to its interaction with viral oncoproteins, pRb is known tobind at least two dozen cellular proteins. J. Wang, Curr Opin Gen. Dev,7:39 (1997); Y. Taya, et al., Trend Biochem. Sci., 22:14 (1997).Included in these is the transcriptional factor E2F-1 which is requiredfor the transcription of certain cellular genes that participate ingrowth control and DNA synthesis (see e.g. W. B. La Thangue, et al.,Biochem. Soc. Trans., 24:54 (1996). It is now clear that E2F is composedof a family of closely related proteins, E2F-1 to -5, and a set ofpartner proteins belonging to the DP family of transcriptional factors(see e.g. J. E. Slansky, et al., Curr. Top. Microbiol. Immunol., 208:1(1996). E2F binds preferentially to the hypophosphorylated form of pRband to several Rb related proteins, including p107 and p130. E2F can bedissociated from pRb and related protein complexes by two mechanisms:hyperphosphoylation of pRb or by the competitive binding with viralproteins. Treatment of cells with TGF-β1 results in the accumulation ofpRb in the hypophosphorylated form. Hypophosphorylated pRb binds E2F-1thus blocking the growth promoting activity of free E2F-1. However,co-expression of viral pRb binding proteins can reverse the TGF-β1mediated arrest of cell growth presumably by displacing E2F-1 from pRb.K. Smith, et al. Virology, 224:184 (1996); K. Zerfass. J. Gen. Virol.76:1815 (1995). Thus, hypophosphorylated pRb binds E2F-1 therebyblocking E2F-1 growth promoting activity whereas phosphorylation of pRbor complexing with viral oncoproteins releases E2F from pRb and leads totranscriptional activation of growth promoting genes.

The association of pRb with transcription factors, such as E2F, occursby interactions at the pocket region and, recently, p107 has also beenshown to exert such a binding profile. Moreover, the pocket region isfound mutated in several human cancers where a lack of function of thepRb protein is thought to be involved in the acquisition of thetransformed phenotype.

There is a need for identification and characterization of new cellulargenes encoding proteins which interact with pRb and which may beinvolved in the regulation of cell growth. The isolation andcharacterization of genes encoding proteins which interact with pRb areparticularly useful in studying mechanisms of cell proliferation and themeans to modulate such activity.

SUMMARY OF THE INVENTION

Applicants have identified nucleotide sequences that encode a novelpolypeptide, designated in the present application as “BOG” (B5TOver-expressed Gene), which exhibits a number of characteristics whichmake it a useful tool for studying cell cycle control and oncogenesis.BOG is a previously undescribed gene whose product binds the pRb tumorsuppressor and is involved in regulating cellular growth. As BOG isshown to bind pRb, a factor known to regulate cellular growth anddevelopment, it is a prototype for molecules that function as endogenousregulators of cellular growth. As such, this novel protein has a varietyof applications in the identification, characterization and regulationof activities associated with cellular regulation as well as processesassociated with oncogenesis.

BOG is over-expressed in a number transformed rat liver epithelial (RLE)cell lines resistant to the growth inhibitor effect of TGF-β1 as well asin primary liver tumors. Over-expression of BOG in non-transformedTGF-β1 sensitive PLE cell lines can confer resistance to the growthinhibitory effect of TGF-β1. Furthermore, continuous over-expression ofBOG in normal RLE cells typically leads to rapid transformation and thetransformed cells can form hepatoblastoma-like tumors when transplantedinto nude mice. Incubation of BOG over-expressing cells with BOGantisense oligonucleotides can restore sensitivity to TGF-β. Moreover,in vivo. BOG typically binds to pRb to form complex that does notcontain E2F-1 and, in vitro. BOG can displace E2F-1 from E2F-1/pRbcomplexes. It is believed that BOG may be important in thetransformation process, due in part, to the capacity of BOG to conferresistance to the growth inhibitory effects of TGF-β1 throughinteraction with pRb and the subsequent displacement of E2F-1.

In one embodiment, the invention provides an isolated nucleic acidmolecule includes DNA which includes nucleotides which encode a BOGpolypeptide. For example, the isolated nucleic acid can include DNAencoding BOG polypeptide having amino acid residues 1 to 173 of Tables1, 5 or 7 or is complementary to such encoding nucleic acid sequence,and remains stably bound to it under at least moderate, and optionally,under high stringency conditions. In another embodiment, the inventionprovides a vector comprising a gene encoding a BOG polypeptide. A hostcell comprising such a vector is also provided. By way of example, thehost cells may be E. coli, yeast or mammalian cells. A process forproducing BOG polypeptides is further provided and comprises culturinghost cells under conditions suitable for expression of BOG. If desired,the BOG may be recovered.

In one embodiment, the invention provides an isolated polypeptidecomprising a BOG fragment, wherein the BOG fragment comprises a pRbbinding motif and a casein kinase II phosphorylation motif. In a favoredembodiment, the isolated polypeptide exhibits BOG like activity and,typically, is capable of binding pRb. In another embodiment, theinvention provides isolated BOG polypeptide. In particular, theinvention provides isolated native sequence BOG polypeptide, which inone embodiment, includes an amino acid sequence comprising residues 1 to173 of Table 1. In a related embodiment, the invention provides chimericmolecules comprising BOG polypeptide fused to a heterologous polypeptideor amino acid sequence. An example of such a chimeric molecule is afactor which includes a BOG fused to a protein such as the maltosebinding protein. In yet another embodiment, the invention provides apolypeptide capable of specifically binding a BOG polypeptide such as anantibody specific for a BOG polypeptide. Optionally, the antibody is amonoclonal antibody.

In other embodiments, the invention provides methods for usingBOG-RELATED polypeptides and nucleic acids for studying and modulatingmechanisms involved in cellular proliferation. In one embodiment, theinvention provides a method of modulating cellular phenotype bycontrolling the level of BOG expression within the cell. In a morespecific embodiment, the invention provides a method of generating atransformed-cellular phenotype by overexpressing BOG. Alternatively, theinvention provides a method of reducing BOG expression via antisenseoligonucleotides in order to effect a cellular phenotype such as TGF-βsensitivity. In a related embodiment, the invention provides methods foreffecting the interaction between pRb and pRb binding proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Northern blot analysis of BOG illustrating the normalexpression pattern in various rat tissues.

FIG. 2(A) shows the yeast two-hybrid system as used to demonstrate theinteraction of Rb and BOG in vivo (Matchmaker Gal4 Two-hybrid System,Clontech). Vectors containing only BOG, Rb, or vector not containinginsert were used as negative controls to demonstrate lack of growth onselective media and lack of β-galactosidase activity. SV40 large T wasused as a positive control to demonstrate interaction with Rb. Growthrates and β-galactosidase activity for BOG and Rb were similar to thoseexhibited by SV40T and Rb.

FIG. 2(B) shows co-immunoprecipitation from whole cell extracts of phi13 and B5T cells were performed with anti-Rb, anti-BOG antibodies andnonspecific preimmune serum. Immunoprecipitates were separated on a 10%SDS-PAGE gel and analyzed by Western blot analysis. Anti-Rb, anti-BOGand anti-E2F-1 antibodies were used to examine the proteins present inthe immunoprecipitated complexes.

FIG. 2(C) shows displacement of E2F-1 from pRb/E2F-1 complexes by BOG.pRb/E2F-1 complexes were immunoprecipitated with either anti-pRb oranti-E2F-1 antibodies. Immunoprecipitates were divided into two samples,and to one sample BOG (a 70 kDa chimeric protein containing BOG fused tothe maltose binding protein) was added. The samples were reprecipitatedwith the same antibody, separated on a 10% SDS-PAGE gel, and the sameblot analyzed by Western blot analysis using anti-Rb, anti-BOG andanti-E2F-1 antibodies.

FIG. 2(D) shows the interaction of retinoblastoma family members andBOG. Antibodies to pRb, p107, p130 and BOG were used to precipitateproteins from whole cell extracts phi 13. The immunoprecipitates wereseparated on a 10% SDS-PAGE gel and analyzed by Western blot analysis todemonstrate the presence of pRb, p107 and p130 in the complexes.

FIG. 3(A) shows colony formation of a heterogeneous population oftransfected RLE and B7 cells expressing BOG. Two RLE cell lines, phi 13and B7, which are untransformed and known to be sensitive to TGF-β weretransfected with BOG or empty vector. After the cells were allowed torecover, they were cultured in the presence of TGF-β1 for 14 days. Mostcells that contained the empty vector control did not grow or diedduring the two weeks in culture. Colonies grow in cells overexpressingBOG were stained with crystal violet.

FIG. 3(B) shows growth curves in the presence of TGF-β1. RLE phi 13cells were transfected with BOG and cell lines overexpressing BOG wereselected. Two clones. BOG-A and BOG-B, representing the two extremes ofBOG expression in these lines were chosen and expanded for furtherexperiments. The effect of overexpression of BOG on the cells ability toproliferate in the presence of TGF-β1 was examined. The effect of TGF-β1on growth is expressed as a percent inhibition of growth of cells grownin the presence of TGF-β1 versus cells grown in normal media for thesame period of time. Values and standard error presented represent twoseparate experiments done in quadruplicate.

FIG. 3(C) shows the effect of BOG expression on the TGF-β1 receptor. Thesame cell lines used to examine growth were expanded and their abilityto bind TGF-β1 was examined. Binding curves were generated using samplesanalyzed in triplicate for each concentration of TGF-β1. Tenfold excessof unlabeled TGF-β1 was used to determine nonspecific binding andsubtracted to obtain specific cpm bound. Saturation curves weregenerated for phi 13. BOG-A and BOG-B with standard error calculatedfrom two separate experiments. To further evaluate the effect of BOG onthe TGF-D receptor expression. Northern blot analysis of TGF-β receptorII was performed (inset). BOG expression also was tested to confirmprevious characterizations of the transgene expression (inset).

FIG. 3(D) shows the restoration of TGF-β1 sensitivity by antisenseoligonucleotides to BOG. In order to decrease the amount of BOG protein,cell lines were incubated with antisense oligonucleotides to BOG.Oligonucleotide alone was not toxic to any of the cell lines asdemonstrated by 100% growth as compared to the cells grown in controlmedia.

FIG. 4(A) shows increased expression of BOG correlates withtransformation. Total RNA was isolated from several untransformed (RLEphi 13 and U1) and transformed (B5T and T1-6) RLE cell lines. (Celllines: phi 13 nontransformed RLE cell line passage 22; B5T, atransformed clone from passage 36; U1=B7, a nontransformed RLE clonepassage 36; T1=AFL-B8, T2=AFL-C2 and T3=AFL-D7 are aflatoxin-transformedRLE cell lines; T4=J2; a v-raf/v-myc transformed RLE cell line; T5=T2and T6=F3611-3, v-raf-transformed RLE cell lines.) 20 μg of total RNAwas analyzed by Northern blot analysis using the 1.9 kb BOG cDNA as aprobe.

FIG. 4(B) shows BOG expression in rat liver tumors. Tumors were derivedutilizing the Solt-Farber carcinogenesis protocol (17) and were analyzedby Northern blot analysis using the 1.9 kb BOG cDNA as a probe. A. B.Roberts, et al., Peptide Growth Factors And Their Receptors, 421-427(1990)

FIG. 4(C) shows tumors from nude mice derived from the BOG-A cell line.Paraffin embedded sections were stained with hematoxylin-eosin (H&E) forhistological evaluation.

FIG. 5 shows the results of a BLAST search and sequence analysis showingthat a Blast search revealed only matches with sequences in ESTdatabase. Search results were obtained by using the open reading frameof rat BOG cDNA. The non-coding region of rat and mouse BOG both containa B-1 like repeat and homology searches with this region produce manymatches. Only the open reading frame of the human BOG sequence isavailable. P(N) value calculated by BLASTN search program with defaultvalues (reference Altschul, S. F. et al.).

FIG. 6(A) shows in vitro translation data and Western Blot datacharacterizing BOG. In vitro translation of rBOG produces a 20 kDAprotein and Western blot analysis identifies a protein of the samemolecular weight.

FIG. 6(B) is a multi-tissue blot analysis showing that rBOG is expressedubiquitously in all tissues analyzed.

FIG. 7 shows a genomic southern blot examining BOG in different speciesand demonstrating that the BOG gene is present in all mammals tested.

FIGS. 8(A) and 8(B) illustrate a chromosomal localization FISH analysisshowing that murine BOG maps to chromosome 2.

FIG. 8(C) is a representational map of the SB6 murine genomic clone.

FIG. 9(A) shows that rat BOG is localized to the nucleus as shown bystaining with polyclonal antibodies.

FIG. 9(B) shows that preincubating the anti-BOG polyclonal antibodieswith BOG antigen inhibits nuclear staining.

FIGS. 10(A) and 10(B) show that the expression of BOG is cell cycleregulated. When RLE and B5T cells-were synchronized at various points inthe cell cycle, both cell lines show a similar pattern of expression ofBOG during the cell cycle, with peak expression being seen during lateG1.

FIG. 11 shows the expression of rat BOG after partial hepotectomy. A ⅔partial hepotectomy was performed on rats to obtain a synchronizedpopulation of primary hepatocytes. The peak expression of BOGcorresponds to late G1-S.

FIG. 12 shows the derivation of RLE cell lines. The RLE cell lines werederived from parental cells isolated from a 10 day old Fisher 344 ratand untransformed and transformed cell lines were derived from thisoriginal population.

FIGS. 13(A)-13(F) shows the sequence and a TESS-String based searchpartial characterization of the murine 5′ genomic BOG DNA sequence (SEQID NO: 11) located approximately 10 nucleotides upstream of the ATGinitiation codon.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMIENTS

1. Definitions

The terms “BOG polypeptidc” and “BOG” when used herein encompass nativesequence BOG and BOG variants (which are further defined herein). TheBOG may be isolated from a variety of sources, such as from human tissuetypes or from another source or prepared by recombinant or syntheticmethods.

The BOG polypeptide, which may be a fragment of a native sequence,contains a retinoblastoma gene product (pRb) binding domain having themotif LXCXE. Typically, the BOG polypeptide also includes at least onecasein kinase II phosphorylation motif that is typically locateddownstream (i.e. closer to the C-terminus of the polypeptide) from thepRb binding domain. Further, the BOG polypeptide may contain a secondcasein kinase II phosphorylation motif that is upstream from the pRbbinding domain.

A “native sequence BOG” is a polypeptide having the same amino acidsequence as an BOG derived from nature. Such native sequence BOG can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence BOG” specifically encompassesnaturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the BOG. In one embodimentof the invention, the native sequence BOG is a mature or full-lengthnative sequence BOG polypeptide comprising amino acids 1 to 173 ofTable 1. Alternatively, the BOG polypeptide comprises amino acidresidues 1 to 173 of Tables 5 or 7.

“BOG variant” means a functionally active BOG as defined below having atleast about 80% amino acid sequence identity with BOG, such as the BOGpolypeptide having the deduced amino acid sequence shown in Tables 1, 5or 7 for a full-length native sequence BOG. Such BOG variants include,for instance, BOG polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the sequence of Tables1, 5 or 7. Ordinarily, a BOG variant will have at least about 80% or 85%amino acid sequence identity with native BOG sequences, more preferablyat least about 90% amino acid sequence identity. Most preferably a BOGvariant will have at least about 95% amino acid sequence identity withnative BOG sequence of Tables 1, 5 or 7. As noted above. BOG variantsinclude a pRb binding domain and typically, one or more casein kinase IIsites. Functionally active BOG variants topically have at least about 50and preferably at least about 100 amino acid residues.

“Percent (%) amino acid sequence identity” with respect to the BOGsequences identified herein is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the BOG sequence, after aligning the sequences in the samereading frame and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared.

“Percent (%) nucleic acid sequence identity” with respect to the BOGsequences identified herein is defined as the percentage of nucleotidesin a candidate sequence that are identical with the nucleotides in theBOG sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising BOG, or a functional fragment thereof, fused to a“tag polypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, or which can beidentified by some other agent, yet is short enough such that it doesnot interfere with activity of the BOG. The tag polypeptide preferablyalso is sufficiently unique so that the antibody does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least six amino acid residues and usually between about 8 toabout 50 amino acid residues (preferably, between about 10 to about 20residues).

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified to a degree sufficient to obtain N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or silver stain. Isolated polypeptide includespolypeptide in situ within recombinant cells, since at least onecomponent of the BOG natural environment will not be present.Ordinarily, however, isolated polypeptide will be prepared by at leastone purification step (referred to herein as an “isolated and purifiedpolypeptide”).

An “isolated” BOG nucleic acid molecule is a nucleic acid molecule thatis identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe BOG nucleic acid. An isolated BOG nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolated BOGnucleic acid molecules therefore are distinguished from the BOG nucleicacid molecule as it exists in natural cells. However, an isolated BOGnucleic acid molecule includes BOG nucleic acid molecules contained incells that ordinarily express BOG where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence: ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking may be accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers may be used inaccordance with conventional practice. “Polynucleotide” and “nucleicacid” refer to single- or double-stranded molecules which may be DNA,comprised of the nucleotide bases A, T, C and G, or RNA, comprised ofthe bases A, U (substitutes for T), C, and G. The polynucleotide mayrepresent a coding strand or its complement. Polynucleotide moleculesmay be identical in sequence to the sequence which is naturallyoccurring or may include alternative codons which encode the same aminoacid as that which is found in the naturally occurring sequence (See.Lewin “Genes V” Oxford University Press Chapter 7, pp. 171-174 (1994)).Furthermore, polynucleotide molecules may include codons which representconservative substitutions of amino acids as described. Thepolynucleotide may represent genomic DNA or cDNA.

“Polypeptide” refers to a molecule comprised of amino acids whichcorrespond to those encoded by a polynucleotide sequence which isnaturally occurring. The polypeptide may include conservativesubstitutions where the naturally occurring amino acid is replaced byone having similar properties, where such conservative substitutions donot alter the function of the polypeptide (See, Lewin “Genes V” OxfordUniversity Press Chapter 1, pp.: 9-13 (1994)).

The term “antibody” is used in the broadest sense and specificallycovers single anti-BOG monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies) and anti-BOG antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

The term “manmmal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. Compositions and Methods of the Invention

A. BOG Nucleic Acids and Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas BOG. In particular, Applicants have identified and isolated genes andcDNA encoding BOG polypeptides, as disclosed in further detail in theExamples below. Using sequence homology searches. Applicants found thatBOG (as shown in Tables 1, 5 and 7) contains the pRb binding motif LXCXEand shares certain amino acid sequence identity with pRb bindingproteins including a number of viral oncoproteins (see Table 2). Asshown in the Examples below, BOG polypeptide was found to bind pRb andto be able to displace other proteins complexed with pRb.

In addition to the full-length native sequence BOG and soluble forms ofBOG described herein, it is contemplated that BOG variants can beprepared. BOG variants can be prepared by introducing appropriatenucleotide changes into the BOG nucleotide sequence, or by synthesis ofthe desired BOG polypeptide. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of theBOG, such as changing the number or position of glycosylation sites oraltering the protein binding characteristics. Variations in the nativefull-length sequence BOG or in various domains of the BOG describedherein, can be made, for example, using any of the techniques andguidelines for conservative and non-conservative mutations set forth,for instance, in U.S. Pat. No. 5,364,934. For example, amino acidsubstitutions at the H and/or D residues of the LHCDE motif arecontemplated, such as conservative substitutions at one or both of theseresidues.

Variations may be a substitution, deletion or insertion of one or morecodons encoding the BOG that results in a change in the amino acidsequence of the BOG as compared with the native sequence BOG. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the BOG. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the BOG with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of 1 to 5 aminoacids. The variation allowed may be determined by systematically makinginsertions, deletions or substitutions of amino acids in the sequenceand testing the resulting variants for activity in any of the in vitroassays described in the Examples below.

In accordance with the practice of this invention. BOG molecules of theinvention can have amino acid substitutions in the amino acid sequenceshown in Tables 1, 5 and 7 (Current Protocols In Molecular Biology,Volume 1, Unit 8. Frederick M. Ausubul et al. eds.; 1995). Suchsubstitutions result in BOG that retains the ability to bind pRb. Theseamino acid substitutions include, but are not necessarily limited to,amino acid substitutions known in the art as “conservative”.

For example, it is a well-established principle of protein chemistrythat certain amino acid substitutions, entitled “conservative amino acidsubstitutions,” can frequently be made in a protein without alteringeither the conformation or the function of the protein. Such changesinclude substituting any of isoleucine (I), valine (V), and leucine (L)for any other of these hydrophobic amino acids; aspartic acid (D) forglutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) andvice versa; and serine (S) for threonine (T) and vice versa. Othersubstitutions can also be considered conservative, depending on theenvironment of the particular amino acid and its role in thethree-dimensional structure of the protein. For example, glycine (G) andalanine (A) can frequently be interchangeable, as can alanine and valine(V).

Methionine (M), which is relatively hydrophobic, can frequently beinterchanged with leucine and isoleucine, and sometimes with valine.Lysine (K) and arginine (R) are frequently interchangeable in locationsin which the significant feature of the amino acid residue is its chargeand the differing pK's of these two amino acid residues are notsignificant. Still other changes can be considered “conservative” inparticular environments.

Variations can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassettemutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selectionmutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)] or other known techniques can be performed on the cloned DNA toproduce the BOG variant DNA. Scanning amino acid analysis can also beemployed to identify one or more amino acids along a contiguoussequence. Among the preferred scanning amino acids are relatively small,neutral amino acids. Such amino acids include alanine, glycine, serine,and cysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia.J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yieldadequate amounts of variant, an isoteric amino acid can be used.

As discussed above, redundancy in the genetic code permits variation inBOG gene sequences. In particular, one skilled in the art will recognizespecific codon preferences by a specific host species and can adapt thedisclosed sequence as preferred for a desired host. For example,preferred codon sequences typically have rare codons (i.e., codonshaving a useage frequency of less than about 20% in known sequences ofthe desired host) replaced with higher frequency codons. Codonpreferences for a specific organism may be calculated, for example, byutilizing codon usage tables available on the INTERNET at the followingaddress: http://www.dna.affrc.go.jp/˜nakamura/codon.html. Nucleotidesequences which have been optimized for a particular host species byreplacing any codons having a useage frequency of less than about 20%are referred to herein as “codon optimized sequences.”

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences which may be deleterious to gene expression. The GC content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. Where possible, the sequence may also be modified to avoidpredicted hairpin secondary mRNA structures. Other useful modificationsinclude the addition of a translational initiation consensus sequence atthe start of the open reading frame, as described in Kozak, Mol. CellBiol., 9:5073-5080 (1989). Nucleotide sequences which have beenoptimized for expression in a given host species by elimination ofspurious polyadenylation sequences, elimination of exon/intron splicingsignals, elimination of transposon-like repeats and/or optimization ofGC content in addition to codon optimization are referred to herein asan “expression enhanced sequence.”

B. Modifications of BOG

Covalent modifications of BOG are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of the BOG with an organic derivatizing agent thatis capable of reacting with selected side chains or the N- or C-terminalresidues of the BOG. Derivatization with bifunctional agents is useful,for instance, for crosslinking BOG to a water-insoluble support matrixor surface for use in the method for purifying anti-BOG antibodies, andvice-versa. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate. In the alternative, BOGcan be joined to a detectable label such as a radioactive isotope suchas I¹²⁵ or P³², an enzyme such as horseradish peroxidase or alkalinephosphatase, a fluorophore such as fluorescein isothiocyanate or achromophore (Current Protocols In Molecular Biology, Volume 2, Units 10,11 and 14, Frederick M. Ausubul et al. eds., 1995: Molecular Cloning, ALaboratory Manual, § 12, Tom Maniatis et al. eds., 2d ed. 1989).

Another type of covalent modification of the BOG polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence BOG, and/oradding one or more glycosylation sites that are not present in thenative sequence BOG. Addition of glycosylation sites to the BOGpolypeptide may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequence BOG(for O-linked glycosylation sites). The BOG amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the BOG polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids. Another means of increasing the number of carbohydrate moietieson the BOG polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

The BOG of the present invention may also be modified in a way to form achimeric molecule comprising BOG fused to another, heterologouspolypeptide or amino acid sequence. In one embodiment, such a chimericmolecule comprises a fusion of the BOG with a tag polypeptide whichprovides an epitope to which an anti-tag antibody can selectively bind.The epitope tag is generally placed at the amino- or carboxyl-terminusof the BOG. The presence of such epitope-tagged forms of the BOG can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the BOG to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag.

In an alternative embodiment, the chimeric molecule may comprise afusion of the BOG with an immunoglobulin or a particular region of animmunoglobulin. The BOG may be fused any one of a variety of knownfusion protein partners that are well known in the art such as maltosebinding protein, LacZ, thioredoxin or an immunoglobulin constant region(Current Protocols In Molecular Biology, Volume 2. Unit 16, Frederick M.Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M.,Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174,561-566). In a preferred embodiment, this fusion partner is a non-BOGbinding molecule so as to prevent difficulties associated withintramolecular interactions. For a bivalent form of the chimericmolecule, such a fusion could be to the Fc region of an IgG molecule.Other fusion proteins and tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an “-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

C. Preparation of BOG

The description below relates primarily to production of BOG byculturing cells transformed or transfected with a vector containing BOGnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare BOG. Forinstance, the BOG sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al. Solid-Phase Peptide Synthesis. W. H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield. J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of theBOG may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length BOG.

1. Isolation of DNA Encoding BOG

DNA encoding BOG may be obtained from a cDNA library prepared fromtissue expressing a BOG mRNA. Accordingly, human BOG DNA can beconveniently obtained from a cDNA library prepared from human tissue.The BOG-encoding gene may also be obtained from a genomic library or byoligonucleotide synthesis. Libraries can be screened with probes (suchas antibodies to the BOG or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Illustrative libraries include mouse kidney cDNA library (mousekidney 5′-stretch cDNA. Clonetech laboratories, Inc.) and human livercDNA library (human liver 5′ stretch plus cDNA, Clonetech Laboratories,Inc.). Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding BOG is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency areprovided in Sambrook et al. supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programswhich employs various algorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for BOG production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample by using lipofectin, CaPO₄ or electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA. 76:3829 (1979). However, other methodsfor introducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations. e.g., polybrene, polornithine, may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example.Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

Suitable host cells for the expression of glycosylated BOG are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9 cells. See e.g.Current Protocols In Molecular Biology, Volume I, Unit 16. Frederick M.Ausubul et al. eds., 1995. Examples of useful mammalian host cell linesinclude rat liver epithelial cells. Hugget, A. C. et. al. Supra. Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding BOG may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general. DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The BOG may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe BOG DNA that is inserted into the vector. The signal sequence may bea prokaryotic signal sequence selected, for example, from the group ofthe alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxinII leaders. For yeast secretion the signal sequence may be, e.g., theyeast invertase leader, alpha factor leader (including Saccharomyces andKluyveromyces “-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2: plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins. e.g.,ampicillin, neomycin, methotrexate, or tetracycline. (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media. e.g. the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the BOGnucleic acid, such as Neomycin, DHFR or thymidine kinase. An appropriatehost cell when wild-type DHFR is employed is the CHO cell line deficientin DHFR activity, prepared and propagated as described by Urlaub et al.,Proc. Nail. Acad. Sci. USA, 77:4216 (1980). A suitable selection genefor use in yeast is the trp1 gene present in the yeast plasmid YRp7[Stinchcomb et al., Nature. 282:39 (1979); Kingsman et al., Gene, 7:141(1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones,Genetics, 85:12 (1977)]. Expression and cloning vectors usually containa promoter operably linked to the BOG nucleic acid sequence to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. Promoters suitable for use with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding BOG.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73.657.

BOG transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovrirus, a retrovirus, hepatitis-B virus and SimianVirus 40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the BOG by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,“-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theBOG coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding BOG. Still other methods, vectors, and hostcells suitable for adaptation to the synthesis of BOG in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting. Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceBOG polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to BOG DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of BOG may be recovered from culture medium or from host celllysates. Cells employed in expression of BOG can be disrupted by variousphysical or chemical means, such as freeze-thaw cycling, sonication,mechanical disruption, or cell lysing agents.

It may be desired to purify BOG from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of theBOG. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular BOG produced.

D. Uses for BOG

Nucleotide sequences for their complement) encoding BOG have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. BOG nucleic acid will also beuseful for the preparation of BOG polypeptides by the recombinanttechniques described herein. BOG polypeptides have various applicationsin the art, including uses for evaluating factors that interact withand/or control pRb function as means for understanding both cell cyclecontrol and oncogenesis. Moreover. BOG genes may introduced into cellsto effect mechanisms mediated b TGF-β as well as processes involved inoncogenesis.

1. Screening Methods Utilizing BOG Nucleic Acids.

The full-length native sequence BOG (Tables 1, 5 and 7) gene, orportions thereof, may be used as hybridization probes for a cDNA libraryto isolate, for instance, still other genes (like those encodingnaturally-occurring variants of BOG or BOG from other species) whichhave a desired sequence identity to the BOG sequences disclosed in Table1, 5 and 7. Optionally, the length of the probes will be about 20 toabout 500 bases. The hybridization probes may be derived from thenucleotide sequence or from genomic sequences including promoters,enhancer elements and introns of native sequence BOG. By way of example,a screening method will comprise isolating the coding region of the BOGgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the BOG gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Nucleotide sequences encoding a BOG can also be used to constructhybridization probes for mapping the gene which encodes that BOG and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

Screening assays can be designed to find lead compounds that mimic thebiological activity of a native BOG or a ligand or receptor for BOG.Such screening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds. The assay-s can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are ell characterized in the art.

2. Modulation of BOG Protein Expression via BOG AntisenseOligonucleotides.

Antisense technology entails the administration of exogenousoligonucleotides which bind to a target polynucleotide located withinthe cells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.BOG. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The BOG antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see. Jack Cohen, supra) which exhibit enhancedcancer cell growth inhibitory action.

S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention may be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol ]-3-one-1,1-dioxide which is a sulfurtransfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990),the disclosures of which are fully incorporated by reference herein.

The BOG antisense oligonucleotides of the present invention may be RNAor DNA which is complementary to and stably hybridizes with the first100 N-terminal codons or last 100 C-terminal codons of the BOG genome orthe corresponding mRNA. While absolute complementarity is not required,high degrees of complementarity are preferred. Use of an oligonucleotidecomplementary to this region allows for the selective hybridization toBOG mRNA and not to mRNA specifying other regulatory subunits of proteinkinase. Preferably, the BOG antisense oligonucleotides of the presentinvention are a 15 to 30-mer fragment of the antisense DNA moleculehaving a sequence which hybridizes to BOG mRNA. Optionally, BOGantisense oligonucleotide is a 30-mer oligonucleotide which iscomplementary to a region in the first 10 N-terminal codons and last 10C-terminal codons of BOG. Alternatively, the antisense molecules aremodified to employ ribozymes in the inhibition of BOG expression. L. A.Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

In one embodiment, the BOG antisense oligonucleotide is coadministeredwith an agent which enhances the uptake of the antisense molecule by thecells. For example, the BOG antisense oligonucleotide may be combinedwith a lipophilic cationic compound which may be in the form ofliposomes. The use of liposomes to introduce nucleotides into cells istaught, for example, in U.S. Pat. Nos. 4,897,355 and 4,394,448, thedisclosures of which are incorporated by reference in their entirety.See also U.S. Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788,4,673,567, 4,247,411, 4,814,270 for general methods of preparingliposomes comprising biological materials. Alternatively, the BOGantisense oligonucleotide may be combined with a lipophilic carrier suchas any one of a number of sterols including cholesterol, cholate anddeoxycholic acid.

In another embodiment, the BOG antisense oligonucleotide may becoadministered with a second agent that is effected by BOG expression.In one embodiment, this second agent is one or more isoforms of TGF-β.In a preferred embodiment, a combination of BOG antisenseoligonucleotides and TGF-β1 are administered to cells which have reducedsensitivity to TGF-β due to BOG overexpression. In this embodiment, acombination of these two molecules may be used to synergistically induceTGF-β1 mediated apoptosis. Methods pertaining to these embodiments arewell known in the art. Zwicker et al., Science 271:1595-1597 (1996);Field et al., Cell 85: 549-561 (1996); Slack et al., J. Cell Bio. 129:779-788 (1995); Hiebert et al., Mol Cell Biol 15: 6864-6874 (1995);White, E. Genes Dev 10: 1-15 (1996); Martin et al., Cell 82:349-352(1995).

The antisense oligonucleotides of the present invention may be preparedaccording to any of the methods that are well known to those ofordinary, skill in the art. Preferably, the antisense oligonucleotidesare prepared by solid phase synthesis. See. Goodchild. J., BioconjugateChemistry, 1:165-167 (1990), for a review of the chemical synthesis ofoligonucleotides. Alternatively, the antisense oligonucleotides can beobtained from a number of companies which specialize in the customsynthesis of oligonucleotides.

3. Use of BOG Nucleic Acids in the Generation of Transgenic Animals.

Nucleic acids which encode BOG or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g. a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g. anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding BOG can be used to clone genomic DNA encodingBOG in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding BOG. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for BOG transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding BOG introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding BOG. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of BOG can be used to construct aBOG “knock out” animal which has a defective or altered gene encodingBOG as a result of homologous recombination between the endogenous geneencoding BOG and altered genomic DNA encoding BOG introduced into anembryonic cell of the animal. For example, cDNA encoding BOG can be usedto clone genomic DNA encoding BOG in accordance with establishedtechniques. A portion of the genomic DNA encoding BOG Can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g. Thomas and Capecchi. Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g. by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g. Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson. ed. (IRL. Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the BOGpolypeptide.

4. Use of BOG Upstream Control Sequences for Evaluating NeoplasticProcesses

The genomic BOG control sequences of the present invention, whetherpositive, negative, or both, may be employed in numerous variouscombinations and organizations to assess the regulation of BOG.Moreover, in the context of multiple unit embodiments and/or inembodiments which incorporate both positive and negative control units,there is no requirement that such units be arranged in an adjacenthead-to-head or head-to-tail construction in that the improvedregulation capability of such multiple units is conferred virtuallyindependent of the location of such multiple sequences with respect toeach other. Moreover, there is no requirement that each unit comprisethe same positive or negative element. All that is required is that suchsequences be located upstream of and sufficiently proximal to atranscription initiation site.

To evaluate BOG regulatory elements in the context of heterologousgenes, one simply obtains the structural gene and locates one or more ofsuch control sequences upstream of a transcription initiation site.Additionally, as is known in the art, it is generally desirable toinclude TATA-box sequences upstream of and proximal to a transcriptioninitiation site of the heterologous structural gene. Such sequences maybe synthesized and inserted in the same manner as the novel controlsequences. Alternatively, one may desire to simply employ the TATAsequences normally associated with the heterologous gene. In any event.TATA sequences are most desirably located between about 20 and 30nucleotides upstream of transcription initiation.

Preferably the heterologous gene is a reporter gene which encodes anenzyme which produces calorimetric or fluorometric change in the hostcell which is detectable by in situ analysis and which is a quantitativeor semi-quantitative function of transcriptional activation. Exemplaryenzymes include esterases, phosphatases, proteases (tissue plasminogenactivator or urokinase) and other enzymes capable of being detected byactivity which generates a chromophore or fluorophore as will be knownto those skilled in the art. A preferred example is E. colibeta-galactosidase. This enzyme produces a color change upon cleavage ofthe indigogenic substrate indolyl-B-D-galactoside by cells bearingbeta-galactosidase (see, e.g., Goring et al., Science, 235:456-458(1987) and Price et al., Proc. Natl. Acad. Sci. U.S.A., 84:156-160(1987)). Thus, this enzyme facilitates automatic plate reader analysisof BOG control sequence mediated expression directly in microtiter wellscontaining transformants treated with candidate activators. Also, sincethe endogenous beta-galactosidase activity in mammalian cells ordinarilyis quite low, the analytic screening system using β-galactosidase is nothampered by host cell background.

Another class of reporter genes which confer detectable characteristicson a host cell are those which encode polypeptides, generally enzymes,which render their transformants resistant against toxins, e.g., the neogene which protects host cells against toxic levels of the antibioticG418; a gene encoding dihydrofolate reductase, which confers resistanceto methotrexate, or the chloramphenicol acetyltransferase (CAT) gene(Osborne et al., Cell, 42:203-212 (1985)). Genes of this class are notpreferred since the phenotype (resistance) does not provide a convenientor rapid quantitative output. Resistance to antibiotic or toxin requiresdays of culture to confirm, or complex assay procedures if other than abiological determination is to be made.

4. Use of BOG Polypeptides in Protein-Protein Interaction Studies.

The illustrative co-immunoprecipitation and Gal4 protein-proteininteraction assays may useful in screening for compounds modulating BOGactivity, or in screening for compounds altering BOG activity in a cell.For example, in proliferating cells, BOG participates with pRb incontrolling the proliferative response of a cell to its environment.Those skilled in the art will understand that binding of a ligand at amolecular binding site can be modulated in a direct manner (e.g., byblocking the site), as ell as altered in an indirect manner (e.g. byconformational changes induced following binding of a second (different)ligand at a distant site). In this regard, it is likely that the bindingsite specificity of BOG for a particular pRb family member (or someother cellular control factor, as discussed below), can be completelyaltered (i.e. to bind a different ligand) by agents that bind at distantsites in the pRb polypeptide. A number of exemplary protocols which maybe used in these studies are known in the art, see e.g. U.S. Pat. No.5,625,031.

It is still further understood that, due to the significance of pRb inthe cell cycle, innate regulator, mechanisms exist in cells forregulating their activity by binding to BOG or to complexes containingBOG. Such regulatory factors can include, at least: a) cofactors thatbind to the complex and exert regulatory action by destabilizing orstabilizing the complex; b) agents that modulate or alter the activityof the complex by inducing conformational changes in the BOGpolypeptides as they are bound together in the complex; c) enzymes thatinactivate one or both members of the complex; and, d) cellular controlfactors (e.g., signal transduction second messengers, transcriptionregulatory factors, and the like) that bind pRb or pRb complexes andmodulate or alter functional activity. Thus, polypeptides such astruncated BOG polypeptides can be constructed that control the activityof the BOG/pRb complexes in the cell by inhibiting or promoting theactivities of such regulatory factors.

Those skilled in the art will recognize that the functional regions ofpRb (including the A/B domains) and BOG are particularly attractivetargets for three-dimensional molecular modeling and for theconstruction of mimetic compounds, e.g., organic chemicals constructedto mimic the three-dimensional interactions between BOG and pRb. Seee.g. J. Wang, Curr Opin Gen. Dev, 7:39 (1997); Y. Taya, et al., TrendBiochem. Sci., 22:14 (1997).

5. Use of BOG Containing Expression Vectors for Modulating CellularPhenotype.

As discussed above. BOG genes can be incorporated into any standardcloning vector. The term “vector” is well understood in the art and issynonymous with the often-used phrase “cloning vehicle”. A suitablevector is a non-chromosomal double-stranded DNA comprising an intactreplicon such that the vector is replicated when placed within aunicellular organism.

Viral vectors include retroviruses, adenoviruses, herpes virus,papovirus, etc. Other suitable vectors include plasmids. Plasmids andretroviruses are generally preferred as vectors.

As discussed in Example 4, pBKCMV-BOG (pBKCMV purchased fromStratagene^(tm)) was constructed and contained the CMV promoter. Thispromoter is suitable for expression of the BOG genes in a wide varietyof cells. However, the CMV promoter is not specifically required fortranscription and expression of the BOG genes. Optionally, one mayreplace the CMV promoter with other knows promoters to improve theefficiency of transcription and expression in particular cells.

The promoter DNA can be amplified using PCR technology whileconcurrently providing restriction sites at the 5′ and 3′ ends of thepromoter DNA. The amplified promoter DNA can then be inserted into acloning vehicle (for example pBKCMV) using conventional endonucleasesand known recombinant DNA technology. Cloning vectors containing thedesired promoter upstream of the 5′ end of BOG genes are constructed inthis manner.

As discussed in Examples 4 and 5, it is possible to influence cellularphenotype using the BOG gene. In this context, cloning vectorscontaining an appropriate promoter and the BOG gene may be constructedusing PCR technology in a manner analogous to the preparation of vectorscontaining exogenous genes as is well known in the art. Cloning vectorscontaining BOG genes are transfected into host cells using knowntransfection processes. Suitable transfection processes includelipofection, electroporation and retrovirus infection. When transfectingcells with BOG, the desired cells are isolated and cultured in suitablemedia.

Transfection of cells using lipofection may conducted according tostandard lipofection procedures. See Felgner et al. 1987. Proc. Natl.Acad. Sci. (USA). 84:7413-7417. In general, liposome-mediated DNAtransfection is accomplished by exposing 1-20 micrograms of plasmid DNAand commercially available liposomes (Bethesda Research Laboratories) inculture medium. The transfected cells are then repeated passaged inculture medium and the desired clones are isolated.

Retrovirus infection may also be accomplished using previously describedprocedures. See for example Miller et al. J. Virol. 62:4337-4345 andHalbert et al. J. Virol. 65:473-378, 1991. In general, plasmid DNA istransfected into a desired packaging cell line such as Psi-2 or othercell lines, using standard calcium phosphate precipitation. Virusesproduced from the Psi-2 cells or equivalent cells are then used toinfect an amphotropic packaging cell line, for example PA317. Virusesproduced by the amphotropic packaging cell line are used to infect thedesired host parent cells of the present invention.

Selection of clones with a modulated phenotype may be undertaken by avariety of protocols that are well known in the art (see e.g. U.S. Pat.No. 5,376,542). In such selections, the cells may be selected for theirability to respond to factors such as TGF-β. Alternatively cells can beselected by their ability to form colonies and grow in soft agar.Moreover, the cells can be selected by their ability to form tumors inanimal models such as in nude mice. After transfection, the desiredclones are selected by culturing in optimal media and repeatedpassaging. Generally, 10-20 passages are required to eliminate spuriouscells and obtain pure clonal cells. Optimal media are selected accordingto the type of parent cell which is utilized. For lymphocytes, RPMImedia is preferred; for fibroblasts, DMEM media is preferred; and forepithelial cells, a serum-free medium such as keratinocyte growth medium(KGM) or SFM (Gibco Company) is preferred.

Selected colonies are then tested to verify the presence of BOG DNA andthe expression of BOG genes. Verification is confirmed by standardSouthern hybridization techniques and immunoprecipitation to determinethe presence or quantity of expressed BOG proteins.

6. Chromosomal Localization.

In Example 8, chromosomal localization of the human and murine BOG genesis described. Chromosomal localization was done by FISH analysis(Stokke, T. et al., Genomics 26: 134-137 (1995)). Murine BOG maps tochromosome 2 and in human to the syntenic region of chromosome 20.

In other embodiments the invention provides diagnostic assays fordetermining chromosomal rearrangement of BOG genes in a cell. Thechromosomal location of BOG genes is conveniently determined inchromosomal smears by in situ hybridization with oligonucleotide probesor cDNA and the like. Translocation of a BOG gene, i.e. from achromosomal location found in a normal cell to a location found in atransformed cell, may contribute to a phenotype of uncontrolled cellgrowth by removing normal transcription regulatory control of eithergene expression of a BOG or RB polypeptide. In the case where therearrangement induces over-expression the cell may acquire a malignant(i.e. uncontrolled) growth phenotype, and in the case where therearrangement induces under-expression the cell may undergo prematuresenescence. Screening cellular samples from individuals for thepotential of BOG chromosomal rearrangement may indicate a relative riskfactor for the possibility of developing cancer.

E. Anti-BOG Antibodies

The present invention further provides anti-BOG antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The BOG antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the BOG polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). Further, polyclonal antibodiesmay be generated commercially, for example by Genemed Synthesis, Inc.using art accepted methods.

2. Monoclonal Antibodies

The BOG antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein. Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the BOG polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies. Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against BOG.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard.Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample. Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal. The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. The monoclonalantibodies may also be made by recombinant DNA methods, such as thosedescribed in U.S. Pat. No. 4,816,567. DNA encoding the monoclonalantibodies of the invention can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of murine antibodies). The hybridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.

Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof particularly. Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized Antibodies

The BOG antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al. J. Mol. Biol., 222:581 (1991)].The techniques of Cole et al., and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1):86-95 (1991)].

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe BOG, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit. Methods for makingbispecific antibodies are known in the art. Traditionally, therecombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy-chain/light-chain pairs, wherethe two heavy chains have different specificities [Milstein and Cuello,Nature, 305:537-539 (1983)]. Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example.Suresh et al., Methods in Enzymology, 121:210 ((1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention.) Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373: EP03089]. It is contemplated that the antibodies may be prepared in Vitrousing known methods in synthetic protein chemists, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

F. Uses for BOG Antibodies

The BOG antibodies of the invention have various utilities. For example,BOG antibodies may be used in diagnostic assays for BOG, e.g., detectingits expression in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982). In addition, BOG or antibodies which recognizeBOG may be used in drug screening assays to identify compounds which actas BOG antagonists or agonists. Antibodies may also be usefultherapeutically either alone, as agents which would act directly tointerfere with the function of BOG or indirectly as targeting agentscapable of delivering a toxin conjugated, for example, pseudomonasexotoxin or radioisotopes, thereto to a desired site.

BOG antibodies also are useful for the affinity purification of BOG fromrecombinant cell culture or natural sources. In this process, theantibodies against BOG are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the BOGto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the BOG, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release theBOG from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va., USA.

Example 1

Isolation of Rat BOG cDNA and Determination of Tissue Expression

Loss of TGF-β1 induced growth inhibition is an early event duringspontaneous transformation of RLE cells. A. C. Hugget, et al., CancerRes., 51:5929 (1991). This resistance to the growth inhibitory effectsof TGF-β1 can clearly be caused by multiple factors. E. R. Barrack,Prostate, 31:61 (1997); R. W. Padgett, et al., Cytokine Growth FactorRev., 8:1 (1997); L. Attisano, et al., Cytokine Growth Factor Rev.,7:327 (1996). However, we had observed a number of the transformed celllines displaying resistance to the growth inhibitory effects of TGF-β1that apparently had “normal” number of TGF-β receptors. A. C. Hugget, etal., Cancer Res., 51:5929 (1991). These observations suggested apost-receptor disruption of the growth inhibitory signal(s) of TGF-β1.To search for endogenous genes which confer resistance to the grouchinhibitory effects of TGF-β1, and in turn may lead to cellulartransformation, subtractive hybridization was done on two RLE cell linesthat were sensitive (RLE phi 13) or insensitive (B5T) or TGF-β. A noveltranscript. BOG (B5T Over-expressed Gene), was identified and shown tobe over-expressed in the B5T, as well as several other transformed RLElines which are resistant to the effects of TGF-β1 (FIG. 4A).

A 1897 bp cDNA clone % as obtained which encoded a protein of 173 aminoacids with a predicted Mr˜19,000 Daltons (Tables 1, 5 and 7). DNAhomology searches of Genbank-revealed BOG to be a unique transcript. BOGmRNA was translated in vitro and the product analyzed by polyacrylamidegel electrophoresis, which confirmed the protein size as 19.3 KDaprotein. We compared the deduced amino acid sequence of BOG with knownproteins in the Swissprot data base. While BOG appeared to be a novelprotein, it contained domains similar to other proteins. The deducedprotein sequence contained a pRb-binding motif LXCXE (Table 1, doubleunderlined) and two casein kinase II phosphorylation sites (Table 1,underline). Alignment of the pRb-binding motif in BOG protein with thatin HPV 16 E7, SV40 large T antigen, adenovirus E1A and Rb-bindingprotein is shown in Table 2. In addition to the pRb-binding region. BOGand HPV16 E7 exhibited 29.6% identity and 7.1% similarity over the wholesequence BOG (Table 3). Expression of BOG in the rat was analyzed usinga multi-tissue northern blot (FIG. 1). Transcript for BOG could bedetected in all tissues but varied in expression levels, demonstratingthe highest expression in the spleen, testis and kidney. The ubiquitousexpression pattern suggests a general rather than cell specific functionfor BOG.

Protocols for the isolation of rat BOG cDNA. Poly(A)+ was isolated fromRLE-13 and B5T cells by using oligo (dt) cellulose, as previouslydescribed (Bradley J E, Bishop G A, St John T, Frelinger J A (1988):Biotechniques 6:114-116. The cDNAs were synthesized using SuperScriptreverse transcriptase (BRL) as recommended by the vendor; added withBstXI adaptor (InVitrogen) and used to produce RLE-13 and B5T cDNAlibraries in pcDNAIneo.

For B5T cDNA enriched subtractive cDNA library construction, B5T andRLE-13 cDNAs were digested with HindIII+XbaI and BamHI+XhoI,respectively and subjected to agarose gel purification. Digested RLE-13cDNA fragments (20 μg) were digested further with AluI-RsaI anddephosphorylated, then hybridized with HindIII+XbaI digested B5T cDNAfragments (0.4 μg). The hybridization mixture was used to construct theB5T cDNA enriched subtractive cDNA library in BluescriptM13 withHindIII-XbaI protruding termini. The enriched library was plated andsingle colonies were isolated. The plasmid from each clone/colony wassequenced, and novel sequences were used to screen a panel of rat tumorsamples and synchronized cells.

The cDNA sequence of rat BOG was determined by double strand sequenceanalysis of a 1.9 kb clone. Sequencing of plasmids was performed usingone of two kits; Dye Terminator Ready Reaction Kits-FS (cat# 402080) orBigDye Termninator Ready Reaction Kits (cat# 4303149), Perkin-ElmerApplied Biosystems. The reactions were purified using Centrisep SpinColumns (cat# CS-901), Princeton Separations. The samples were analyzedon a 377 ABI Prism DNA sequencer, Perkin-Elmer Applied Biosystems. Thepredicted amino acid sequence was derived using PCGENE and indicatedbelow with single letter code.

Protocols for BOG Northern blot analysis. Poly (A)+ RNA was isolated andselected using oligo (dt) cellulose (Bradley J E, Bishop G A, St John T,Frelinger J A (1988), Biotechniques 6:114-116.), and 5 μg fractionatedunder denaturing conditions by electrophoresis through 1.2%agarose-formaldehyde gel, transferred to nitrocellulose and hybridizedwith a 500 bp fragment of BOG corresponding to the open reading frame.All blots were rehybridized with a probe to actin to ensure equalloading and integrity of the mRNA samples.

Example 2

Assessing BOG and pRb Interaction Using a Yeast Two-Hybrid System.

As stated previously, BOG contains the LXCXE pRb-binding motif found inmany of the Rb binding proteins. To test the interaction between BOG andpRb, we employed the yeast two-hybrid system. The appropriate plasmidconstructs were sequentially transformed into yeast reporter strainsGAL1 promoter activity was analyzed by growth on plates lackinghistidine. CYC1 promoter activity also was analyzed by induction of lacZby staining clones on X-gal containing media (FIG. 2A). As a positivecontrol for both promoters SV40 large T antigen (pTD1) interaction withpRb (pGBT9/Rb) was used. The intensity of X-gal staining for Rb and BOGas compared to pRb and SV40 large T suggests that their degree ofinteraction is comparable.

Protocols involving the Gal4 two-hybrid system. The yeast two-hybridsystem was used to demonstrate the interaction of Rb and BOG in vivo(Matchmaker Gal4 Two-hybrid System, Clontech). The complete open readingframe of BOG was cloned into pGAD424, and a fragment of Rb, the 1.9 kbBsmHI-SalI fragment of Rb from pASRB2 was cloned into pGBT9. All otherplasmids, SV40T (pTD1) GAL4 (pCL1) were obtained from Clontech's twohybrid system. The plasmids were sequentially transformed into yeaststrain HF7c and picked by growth on selective media. The appropriateclones were streaked onto filters and assayed for β-galactosidaseactivity. Yeast clones with plasmids containing only Rb, BOG, or bothexpression vectors without insert served as negative controls, while theinteraction of SV40T and Rb, and wild type GAL4 served as positivecontrols. The yeast Matchmaker Gal4 Two-hybrid System (cat# K1605-1),Clontech Laboratories, Inc., contains the vectors pGAD424, pGBT9, pTD1and pCL1.

Example 3

Assessment of BOG, E2F-1 and pRb Interaction via CoimmunoprecipitationStudies. To confirm the interaction of pRb and BOG, whole cell lysateswere subjected to co-immunoprecipitation with either anti-pRb,anti-E2F-1 or anti-BOG antibodies and analyzed by Western blot (FIG.2B). From cells over-expressing BOG, pRb could be precipitated with bothanti-BOG and anti-Rb. However when the blot is stripped and re-analyzedwith anti-E2F-1. E2F-1 could be detected only in the samplesprecipitated with anti-Rb. This indicates that the pRb associated withBOG is not bound to E2F-1, suggesting that BOG competes and coulddisplace E2F-1 from pRb. This model is supported by the analysis of theB5T cells which over-express BOG. When one compares the amount of pRbprecipitated from RLE and B5T cells by anti-pRb antibodies, similaramounts of pRb are present in the whole cell extracts (FIG. 2B).However, in the presence of greater amounts of BOG as in the B5T cells,the amount of E2F-1 bound in the pRb complexes is dramatically reduced.An important implication of this observation is that overexpression ofBOG may be needed to displace E2F-1 from pRb.

To test directly if BOG could displace E2F-1 from pRb/E2F-1 complexes,non-transformed RLE cells were treated with TGF-β1 for 24 hours to allowfor synchronization of the cells in late G1, and the accumulation ofRb/E2F-1 complexes. The pRb/E2F-1 complexes were immunoprecipitated witheither anti-Rb or anti-E2F-1 antibodies, and incubated with the fusionprotein BOG-MBP. The lysates were precipitated again and analyzed byWestern blot analysis (FIG. 2C). These results show clearly that BOGbinds to pRb and can compete with and displace E2F-1 bound to pRb.

It has also been reported that viral genes that contain the LXCXE pRbbinding consensus sequence can interact with the other retinoblastomagene family members. P. Whyte, et al., Nature, 334:124 (1988); W. H.Lee, et al., Science, 235:1394 (1987); R. Bernards, et al., Proc. Natl.Acad. Sci. USA, 86:6474 (1989). Both p130 and p107 share homology topRb, containing highly conserved domains including H1, H2 and the largepocket subdomains A and B. To evaluate if BOG also interacts with theseproteins, whole cell lysates were prepared from RLE cells andco-immunoprecipitation experiments were performed with anti-BOG,anti-pRb, anti-p130 and anti-p107 antibodies. As illustrated in FIG. 2D,BOG is present in complexes with all the Rb family members. Thissuggests that overexpression of BOG may affect the regulation p107 andp130, as well as pRb. Changes in the regulation of p107 and p130 may bean important contributing factor in the rapid transformation seen in theRLE cell line, however, the specific effect of BOG overexpression onthese Rb family members needs to be further investigated.

Protocols for the precipitation of BOG from whole cell extracts.Co-immunoprecipitation from whole cell extracts of phi 13 cells teasperformed with anti-Rb 1F8 (Santa Cruz Biotechnology), anti-BOGantibodies and nonspecific preimmune serum.

Immunoprecipitates were separated on a 10% SDS-PAGE gel and analyzed byWestern blot analysis using anti-Rb, anti-BOG and anti-E2F-1 antibodies(Santa Cruz Biotechnology). To assay the ability of BOG to displaceE2F-1 from the pRb complex, a chimeric protein was constructed using theProtein and Purification System (New England Biolabs. Inc.). The pMAL-P2vector used produced a protein in Which BOG is fused to thecarboxy-terminal of the maltose binding protein (MBP). The chimericprotein was purified using an amylose resin and tested for its abilityto precipitate pRb from whole cell lysates. Protein preparations capableof precipitating pRb were used for displacement experiments. Thepurified IMIBP without BOG did not precipitate any proteins from wholecell lysates under similar conditions. Displacement of E2F-1 frompRb/E2F-1 complexes by BOG were performed with RLE phi 13 cells treatedwith TGF-β1 (500 pmol) for 24 hours to maximize pRb/E2F-1 complexformation. Cells were harvested and whole cell extracts wereimmunoprecipitated with anti-Rb and anti-E2F-1 antibodies (Santa CruzBiotechnology). Immunocomplexes were aliquoted into two factions, and toone fraction the chimeric BOG-MBP was added the second fraction servedas a control and received only buffer. Both fractions were allowed toincubate for 1 hour. The mixtures were immunoprecipitated again the sameantibodies as before. The immunoprecipitates were separated on a 10%SDS-PAGE gel and analyzed by Western blot analysis using anti-Rb,anti-BOG, and (¹²⁵I) anti-E2F-1 antibodies. Immunoprecipitation for p107and p130 (anti-p107 C18 and anti-p130 C20; Santa Cruz Biotechnology) wasperformed on whole cell extracts of RLE phi 13.

For the experiments involving PAGE electrophoresis and Western blotting,SDS-PAGE and electro-transfer blotting was performed using XCell IIMini-Cell (Dual Slab) & Blot Module (cat# EI9002), Novex. Proteins wereseparated on 10% Tricine gels 1.0 mm/10 well (ca# EC6675) using theappropriate buffer systems; Tricine SDS running buffer (10×) (cat#LC1675) and Tricine Sample Buffer (2×) (cat# LC 1676), Novex. Gels wereelectro-transferred to pre-cut Nitrocellulose (cat# LC2001), Novex. Invitro translation was performed on the rat cDNA using TnT T7 quickCoupled Transcription/Translation System (cat# L1171), Promega.

Example 4

Evaluation of Cellular Phenotype as a Function of BOG Expression.

Previous studies in our laboratory indicated that spontaneoustransformation of RLE cells was accompanied by acquisition of resistanceto TGF-β1 mediated growth inhibition and suggested that the resistanceto TGF-β1 was an important and possibly essential step in thespontaneous transformation process. A. C. Hugget, et al. Cancer Res.,51:5929 (1991). We reasoned that over-expression of BOG, acting bydisplacing E2F-1 from pRb might, at least in part, account for loss ofTGF-β1 growth inhibition observed during spontaneous transformation ofthe RLE cells. To test this hypothesis constitutive over-expression ofBOG was established in the non-transformed RLE and B7 cell lines. Thecells transfected with either pcDNA-BOG or control plasmid pcDNA, weresubjected to neomycin selection, and pools of neomycin-resistant RLE,RLE/BOG, B7 and B7/BOG cells were generated. The four pools were thencultured in the presence of 0.5 ng/ml TGF-β1. The presence of pcDNA-BOGplasmid in RLE/BOG and B7/BOG cells significantly decreased thesensitivity of these cells to growth inhibition by TGF-D growth (FIG.3A).

We also compared the TGF-β1 sensitivity in single cell clonestransfected with either control plasmid pBKCMV or with pBKCMV-BOGcontaining expression plasmid pBKCMV-BOG. Expression of BOG in theseclones was 3 to 7.5 fold higher than in the TGF-β1 sensitive RLE cells(FIG. 3C inset). Over-expression of BOG did not-significantly affecteither the binding of TGF-β1 to the cell surface receptors of TGF βreceptor II mRNA expression (FIG. 3C).

To address the possible linkage between over-expression of BOG andtransformation further, we analyzed expression of BOG in varioustransformed and non-transformed cells lines derived from the RLE cells(FIG. 4A). Expression of BOG in non-transformed RLE (lane 1) and B7 celllines U1 (lane 3) was very low, while higher expression levels weredetected in all transformed cell lines except T5 (lane 8), a cell linetransformed by v-raf. A. C. Hugget, et al., Cancer Res., 51:5929 (1991).All the cell lines examined exhibited a similar doubling time, and sinceRNA was extracted from the cells during log phase growth, mitotic indexalone can not account for the increased expression of BOG in thetransformed cell lines.

However, since the T5 line, one of the more aggressive transformed RLEcell lines, did not demonstrate increased expression of BOG, suggestingthe overexpression of BOG is not an absolute requirement fortransformation of the RLE cells. To evaluate the relationship betweenBOG expression and transformation in vitro hepatocellular carcinomaswere chemically induced in male rats. A. B. Roberts, et al., In PeptideGrowth Factors And Their Receptors, 421-427 (1990). Six tumors wereanalyzed for BOG expression, and all displayed elevated expression ascompared to normal rat liver tissue (FIG. 4B). It is important torestate here that one of the early events in the transformation processin the liver is loss of sensitivity of TGF-β. While the increasedexpression of BOG in the tumors may be due in part to the increasedmitotic activity in the tumor, an increased expression of BOG in all thetumors may reflect the importance of BOG in the selection of the clonalphenotype that maintained a growth advantage during neoplasticdevelopment in the liver.

Finally, we evaluated the impact of constitutive over-expression of BOGon the rate of spontaneous transformation of RLE cells. Cellsover-expressing BOG exhibited aberrant morphology 5 to 6 passages afterBOG transfection, and by passage 9, 80% of the cell population wascomprised of morphologically transformed cells, as shown by smaller cellsize with little cytoplasm and enlarged nucleus compared to the originalearly passage. This is in stark contrast to the parental RLE cells thatrequire a minimum of 35-40 passages to begin displaying the transformedphenotype. A. C. Hugget, et al. Cancer Res., 51:5929 (1991). WhenRLE/BOG cells were kept at a confluent stage, small colonies arosedemonstrating the ability of these cells to grow in an anchorageindependent manner. Further confirmation of the transformed phenotypewas demonstrated by the capacity of RLE/BOG cells to grow in a soft agarand to form tumors in nude mice (FIG. 4C). Tumor phenotype wasconsistent with that of a highly vascular hepatoblastoma-embryonal type,similar to tumors formed by RLE cells transformed by v-raf/v-myc. S.Garfield, et al., Mol. Carcinog, 1:189 (1988). Southern blot analysis ofRLE and B5T indicate amplification or rearrangement of the BOG gene isnot the source of increased expression in these cell lines.

The cell lines used herein were first described in: Hugget. A. C. et.al. Development of resistance to the growth inhibitory effects oftransforming growth factor beta 1 during the spontaneous transformationof rat liver epithelial cells. Cancer Res. 51, 5929 (1991), andGarfield. S. et. al. Neoplastic transformation and lineage switching ofrat liver epithelial cells by retrovirus-associated oncogenes. Mol.Carcinog. 1, 189 (1988).

Protocols for Evaluating Constitutive Over-Expression of BOG in RLE CellLines. Colony Formation of Heterogeneous Population of Transfected RLEand B7 Cells Expressing BOG.

The cDNA HindIII-XbaI cDNA fragment from subtractive clone inpBluescriptM13 was subcloned into the mammalian expression vectorpCDNAIneo (Invitrogen), which contains the CMV promoter and a neomycinselectable marker. The expression plasmid pCDNAI-BOG and the controlvector pCDNACMV were transfected into phi 13 and B7 cells by lipofectin(Gibco BRL). After a 3 weeks selection in Ham's F-12 media containingG418 400 log activity/ml (726 μg/ml) (Sigma), colonies were harvested asmixed colonies and maintained in selective media. For the colonyformation assay, 10⁵ cells for phi 13CMV (phi 13 transfected with theempty vector pCDNAIneo as a control) and phi 13 BOG (phi 13 transfectedwith expression plasmid pCDNAI-BOG) or 5×10⁴ cells for B7CMV (B7 cellswith pCDNAIneo) and B7BOG (B7 cells with pCDNAI-BOG) were plated, into100 mm diameter dishes. The cells were allowed to attach and mediacontaining 0.5 ng/ml TGF-β1 (R&D Systems) was added into the dishes 18hours after plating. The medium containing 0.5 ng/ml TGF-β1 was changedevery 3 days for 15 days on all plates. After 15 days of treatment thecells were fixed and the dishes were stained using crystal violet. Toassess the effects of BOG on growth curves in the presence of TGF-β1,the open reading frame of BOG was subcloned into the mammalianexpression vector pBKCMV (Stratagene), which contains the CMV promoterand neomycin selectable marker. Phi 13 cells were transfected withlipofectin, and grown under selection in G418 until single cell cloneswere visible. Several clones were picked and expanded there passages inG418 containing media. The expression level of BOG was checked byNorthern blot analysis in all clones and representative clones (BOG-1and BOG-B) were used for further experiments. To analyze for TGF-β1growth inhibition, normal phi 13, BOG-A and BOG-B cells were plated at5×10³ into 96 well plates.

After 6 hours when the cells where attached, the media was chanced toHam's F-12 media containing either 0, 100 pg/ml or 1000 pg/ml of TGF-β1.The cultures were maintained for 72 hours, after which time a cellproliferation assay was performed (Promega). The effect of TGF-β1 ongrowth is expressed as a percent inhibition of growth of cells grown inthe presence of TGF-β1 versus cells grown in normal media for the sameperiod of time. To examine the effect of BOG expression on the TGF-β1receptor, phi 13, BOG-A and BOG-B cells were plated in 24-well platesand grown to 90% confluency. TGF-0 and anti-E2F-1 were both iodinated bythe method of Sambrook J, Fritsch E F. Maniatis T (1989): “MolecularCloning: A Laboratory Manual.” 2^(nd) Ed., Cold Spring Harbor, N.Y.:ColdSpring Harbor Laboratory. Cells were analyzed in triplicate plates foreach concentration of TGF-β1. Before the binding assay was performed thecells were washed once with binding buffer (Hanks' solution containing20 mM HEPES pH 7.0) and equilibrated in the same buffer for 30 min at 4°C. Ice-cold binding buffer containing increasing concentrations of¹²⁵I-TGF-β1 (0 to 500 pM ¹²⁵I-TGF-β1) was added and the preparation wasincubated at 4° C. for 1 hour. Tenfold excess of unlabeled TGF-β1 wasused to determine nonspecific binding and subtracted to obtain specificcpm bound. Cultures were washed 3 times with ice-cold binding buffer,and the bound and unbound (media and first two washes) ¹²¹I-TGF-β1 wasmeasured in a LKB Rackgamma II gamma counter. Saturation curves weregenerated for phi 13, BOG-A and BOG-B with standard error calculatedfrom two separate experiments.

Protocols for assessing colony formation in agar and tumor formation innude mice. Methods for growth in soft agar and transplantationexperiments were followed as described in Hugget, A. C. et. al.Development of resistance to the growth inhibitory effects oftransforming growth factor beta 1 during the spontaneous transformationof rat liver epithelial cells. Cancer Res. 51, 5929 (1991), andGarfield, S. et. al. Neoplastic transformation and lineage switching ofrat liver epithelial cells by retrovirus-associated oncogenes. Mol.Carcinog. 1, 189 (1988). Tumors in nude mice were derived from BOG-Acell line. Morphologically transformed BOG-A (passage 14) cells (106)cells were transplanted into nude mice. Visible tumors developed withintwo weeks, at which time the animal was sacrificed and tumors dissected.Tumors were fixed in 10% phosphate buffered (pH 7.4) formalin, embeddedin paraffin and sections stained with hematoxylin-eosin (H&E) forhistological evaluation.

Example 5

Effects of BOG Antisense Oligonucleotides on BOG Expression and TGF-βSensitivity.

As discussed above, the growth inhibitory effects of TGF-β1 werecompletely blocked by BOG over-expression (FIG. 3B). To examine if wecould restore sensitivity to TGF-β1 by decreasing the amount of BOG inthe cell, we incubated cells with antisense oligonucleotides to BOG. Inthe cell line BOG-A, that had a 3 fold overexpression of BOG, incubationwith antisense oligonucleotides restored sensitivity to TGF-β1 to alevel similar to that of the parental RLE cells (FIG. 3D). Moreover,sensitivity to TGF-β1 could also be restored in the B5T cell line, atransformed cell line that was less sensitive to TGF-β1, but is known tocontain the full compliment of TGF-β receptors. This suggests that oneof the primary causes for loss of sensitivity to TGF-β1 in these celllines is due to the overexpression of BOG. One implication of thisobservation is that sensitivity to TGF-β may be restored in transformedcells which still maintain signaling through the TGF-β pathway, throughmethods that decrease the expression of BOG. Furthermore, sinceoverexpression of BOG decreases sensitivity to TGF-β even in thepresence of an intact TGF-β signaling pathway, BOG may be an earlyindicator or selection for cells destined to become transformed in vivo.

Protocols for the inhibition of BOG by anti-sense oligonucleotides.Phosphothionate-modified antisense oligonucleotides corresponding to the5′ end (AATCACTGCCTTGGTAGGGGACACCATTAA) (SEQ ID NO:12) and the 3′ end ofthe one reading frame (TCAGTTCATGGAACTCTGTGTTCTGAAAGTGAC) (SEQ ID NO:13)of BOG were synthesized and purified by OPC column (BiosemeBiotechnology, Inc.). Cells were plated at 50% confluency and allowed toattach in Ham-F12 media containing 10% Fetal Bobine Serum (FBS). Oncethe cells were attached, they were washed once with DPBS and mediacontaining antisense oligonucleotides (340. mu.g/ml each) and/orTGF-.beta. 1 (250 pg/ml) was added to the cells, and the cultures wereallowed to grow for 48 hours. Cell growth was assayed using the CellTiter 96 Non-radioactive Cell Proliferation Assay (CellTiter 96Non-Radioactive Cell Proliferation Assay (cat#G4000). PromegaCorporation). The concentration of TGF-β was determined by art acceptedmethods. In particular, the dose response of the cells to TGF-β waspreviously determined (Hugget, A. C. et. al.). Development of resistanceto the growth inhibitory effects of transforming growth factor beta 1during the spontaneous transformation of rat liver epithelial cells.Cancer Res. 51, 5929 (1991)). A dose of 0.5 ng/ml TGF-β was chosenbecause it was an effective dose for the cytostatic effect in all celllines analyzed.

Example 6

Generation of a BOG-MBP Fusion Protein.

A chimeric protein (BOG-MBP) was constructed using the Protein Fusionand Purification System (New England Biolabs, Inc.). PCR protocols wereutilized to generate a BOG polynucleotide having restrictionendonuclease sites that allowed the complete open reading frame to belinked to the maltose binding protein (MBP). See e.g. Current ProtocolsIn Molecular Biology, Volume I, Unit 17. Frederick M. Ausubul et al.eds. 1995. The pMAL-P2 vector used produced a protein in which thecomplete rBOG protein is fused to the carboxy-terminal of MBP(pMAL-P2-BOG). The chimeric protein was purified using an amylose resinand tested for its ability to precipitate pRb from whole cell lysates.The DNA sequence encoding an extracellular region of the BOGpolypeptide.

Example 7

Preparation of Antibodies that Bind BOG.

Polyclonal antibodies to BOG was generated commercially by GenemedSynthesis, Inc. using art accepted methods. Initially, the rabbits wereimmunized with the fusion protein (BOG-MBP) and subsequent booster shotswere done with purified 20 kDa BOG. The final serum generated recognizedboth BOG and MBP.

The following example illustrates preparation of monoclonal antibodieswhich can specifically bind BOG.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified BOG, fusion proteins containing BOG, andcells expressing recombinant BOG on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the BOG immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch. Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectBOG antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of BOG. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids. The hybridoma cells will be screened in an ELISA forreactivity against BOG. Determination of “positive” hybridoma cellssecreting the desired monoclonal antibodies against BOG is within theskill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-BOGmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 8

Isolation and Chromosomal Localization of Human and Murine BOG DNASequences. DNA comprising the coding sequence of BOG was employed foruse as both primers and probes to screen for homologous DNAs (such asthose encoding naturally-occurring variants of BOG) in human tissue cDNAlibraries, human tissue genomic libraries. Analysis of cDNA librariesfrom human and mouse indicate that BOG is a highly conserved gene inboth these species.

The coding region of the human cDNA was isolated from a human liver cDNAlibrary (human liver 5′ stretch plus cDNA, Clonetech Laboratories, Inc.)using PCR. See Table 4. Primers were designed to the 5′(ATGGTCTCTCCTAGC) (SEQ ID NO:14) and 3′ (GAGTTCCATGAAC) (SEQ ID NO: 15)portions of the open reading frame of mouse and rat BOG, and PCR wasdone using PCR SuperMix (Gibco, BRL Life Technologies) using companyrecommended conditions. The predicted 500 bp PCR fragment was clonedinto the TA vector pCR2.1 (In Vitrogen.TM.), for further analysis(pCR2.1huBOG). Five clones were isolated and double strand sequencedobtained to confirm hBOG sequence.

The mouse cDNA clone was isolated from a mouse kidney cDNA library(mouse kidney 5′-stretch cDNA, Clonetech laboratories, Inc.). See Table6. Phage were plated at 5×10⁵ pfu/150 mm plate and duplicate filterswere screened using a 500 bp fragment corresponding to the coding regionof rBOG. Seven plaques were identified and plaque purified. The insertfrom these lambda clones were subcloned into the pBluescript vector(Stratagene™) for further analysis (pBSλmBOG). All clones were doublestrand sequenced and used to confirm overall sequence of mBOG.

Human and murine genomic clones were isolated by screening high densityfilters of BAC genomic libraries supplied by Genome Systems, Inc. SeeTables 8 and 9.

Chromosomal localization was done by FISH analysis (Stokke, T., Collins,C., Kuo, W. Kowbel, D., Shadrvan, F., Tanner, M., Kallionienmi, A.,Kallioniemi, O., Pinkel, D., Deaven, L., and Gray, J; A physical map ofchromosome 20 established using fluorescent in situ hybridization (FISH)and digital image analysis. Genomics 26: 134-137 (1995). BOG maps tomurine chromosome 2. In humans. BOG maps to the syntenic region ofchromosome 20. See FIGS. 8A and 8B.

Example 9

Evaluation of BOG Expression During Cell Cycling. All cell lines asmentioned above were routinely maintained in Ham's F-12 mediumsupplemented with 10% defined fetal bovine serum (Biofluids, Rockville,Md.) and 50 ug/ml gentamicin (GIBCO, Grand Island, N.Y.) at 37° C. in ahumidified atmosphere of 5% CO2 and 95% air. Cells were grown in 162 cm²plastic flasks and passaged at a ratio 1:8 every 4 days using 0.1%trypsin solution in versene (Biofluids) for cell detachment.

Cells were plated in 162 cm² plastic flasks at 2×10⁶ cells/flask forRLE-13 cells and at 3×10⁶/flask for B5T cells. Synchronization was donefollowing 3 types of protocols. For synchronization by Lovastatintreatment, medium was removed 24-36 hours after the initial plating andreplaced with fresh medium containing Lovastatin at 10 uM for RLE-13cells and 30 uM for B5T cells. To release cells from this block, after24 hour incubation (time 0 hour) the medium was removed and replacedwith fresh medium containing mevalonic acid at a concentration 100 timesthe Lovastatin concentration used. For experiments measuring effects ofTGF-β1 on BOG expression during cell cycle TGF-β1 (250 pg/ml) was addedin the fresh medium. Cells were harvested at the indicated times using0.1% trypsin solution in versene for flow cytometer analysis Forsynchronization by serum deprivation, cells were incubated with Ham'sF12 medium containing 0.2% defined fetal bovine serum for 72 hours. Attime 0 hours, the low serum medium was removed and the cells werestimulated by addition of complete medium containing 10% serum. Forsynchronization by mimosine; aphidicolin and nocadazole, medium wasremoved and replaced with fresh medium containing 200 uM mimosine(Aldrich), 5 ug/ml aphidicolin (Sigma) or 400 ng/ml nocadazole(Janssen). Cells were harvested by trypsinization and centrifugation.For RNA isolation, cells were lysed in 4M guanidine thiocyanatesolution. For flow cytometer analysis, cells were fixed by dehydrationin 70% ethanol and stored at 4° C. prior to analysis. Cells were thenwashed once with ice-cold PBS, treated with 500 units of RNase A(Boehringer Mannheim) per ml for 15′ at 37° C. Cellular DNA was stainedwith 50 ug of propidium iodide per ml for a minimum of 30 minutes priorto flow cytometer analysis. Cell cycle determination was performed usinga Becton-Dickinson Fluorescence-Activated Cell Analyzer and data werecollected on the FL2 channel using a 640 nm band pass filter. Resultsrepresent a minimum of 10,000 cells assayed for each determination.Results of these experiments demonstrate that transcription of BOG iscell-cycle regulated, having high expression in Go, decreasing in earlyG1, and increasing and peaking in expression late G1/S. See FIGS. 10Aand 10B.

Protocols to establish the nuclear localization of BOG polypeptides wereperformed following art accepted immunohistochemical methods usinganti-BOG antibodies. See FIGS. 9A and 9B.

Table 1 shows the cDNA (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2)sequences of rat BOG as determined by double strand sequence analysis ofa 1.9 kb clone. The potential Rb binding domain is indicated by theunderline and the two putative casein kinase II phosphorylation sites byboldface type. GTCTCACGTC TGCATTA ATG GTG TCC CCT ACC AAG GCA GTG ATTGTT CCT 50                   MET VAL SER PRO THR LYS ALA VAL ILE VAL PRO                    1               5                   10 GGG AAC GGAGGC GGG GAT GTG GCC ACC CAC GGC TGG TAC GGC TGG GTG 98 GLY ASN GLY GLYGLY ASP VAL ALA THR HIS GLY TRP TYR GLY TRP VAL             15                  20                  25 AGA AAG GGG CTGGAG CAG ATT CCT GGT TTC CAG TGT TTG GCT AAA AAC 146 ARG LYS GLY LEU GLUGLN ILE PRO GLY PHE GLN CYS LEU ALA LYS ASN         30                  35                  40 ATG CCT GAC CCA ATTACC GCT CGA GAG AGC ATC TGG CTG CCC TTC ATG 194 MET PRO ASP PRO ILE THRALA ARG GLU SER ILE TRP LEU PRO PHE MET     45                  50                  55 GAG ACA GAA CTG CAC TGTGAT GAG AAG ACC ATC ATC ATA GGC CAC AGT 242 GLU THR GLULEU HIS CYS ASP GLU LYS THR ILE ILE ILE GLY HIS SER 60                  65                  70                  75 TCC GGGGCC ATC GCA GCC ATG AGG TAT GCA GAG ACA CAT CAG GTA TAC 290 SER GLY ALAILE ALA ALA MET ARG TYR ALA GLU THR HIS GLN VAL TYR                 80                  85                  90 GCT CTC ATATTG GTG TCT GCA TAC ACA TCA GAC TTG GGA GAT GAA AAT 338 ALA LEU ILE LEUVAL SER ALA TYR THR SER ASP LEU GLY ASP GLU ASN             95                 100                 105 GAG CGT GCA AGTGGG TAC TTC AGC CGC CCC TGG CAG TGG GAG AAG ATC 386 GLU ARG ALA SER GLYTYR PHE SER ARG PRO TRP GLN TRP GLU LYS ILE        110                 115                 120 AAG GCC AAC TGC CCTCAC ATT ATA CAG TTT GGC TCT ACT GAT GAC CCC 434 LYS ALA ASN CYS PRO HISILE ILE GLN PHE GLY SER THR ASP ASP PRO    125                 130                 135 TTC CTT CCA TGG AAG GAACAA CAA GAA GTG GCA GAT AGC TGG ACG CCA 482 PHE LEU PRO TRP LYS GLU GLNGLN GLU VAL ALA ASP SER TRP THR PRO140                 145                 150                 155 AAC TGTACA AAT TCA CTG ACC GTG GTC ACT TTC AGA ACA CAG AGT TCC 530 ASN CYS THRASN SER LEU THR VAL VAL THR PHE ARG THR GLN SER SER                160                 165                 170 ATG AACTGATTAGAGT GGTGAAGTCT ATGCTGACTC CTGCTCTGTA ACACGCCAGG 586 MET ASNATGGGGTAGA AGAGTAACAG CCGCTACCCT CACACAGCTT AGACATGGAC GTCCGTCCAG 646TTAGACTACA GAAGTGTCTG AGCAACAAAC CCATTTGAAC ACTCACACTG AGTTAGTAGC 706ACTTCCAGTT CCCACAGAGC TTAAATCTCC CCAAAAGCTA CTAGCTACAG CAGTATGTTT 766CCTGTTTGAT AAGAGACAGG TTTTTTATTT TTAAGCTATC CTGTTGATGC AAAGAGAGTT 826AAGTCAGAAG AATCCAGAAC TTGACATAGA CCTGGTTGTG TGTCCCTGTA ATCATTTCAG 886AAAGCAGGGT CAGGAGGGAA GGCTATCTTG ACCCTGTCTC AGAAAGAGTG AGCAAGAAGA 946TGACCCAGGT CTCCTGAGGC TCATTCCAAA TTATAACTCA CTATGTTTAG CAAGATGTGT 1006TCCACTTCTG AGACCCCAGT ACTTGGAAAA CTGAGGAAGG TTCATCTTGA GTTTGAGACC 1066TTGTCTAGGC TAAGTAAACC CTGTCTCAAA ACAACAACAA AAACAGGTTT TCATTCAACT 1126TATATGACTG ACACTTTCCA TTTGTAATAA AAAATTTTCT CTTACTGGGG AAATGAAAAC 1186ACGATTCAAG GTCCAGAATG TTGTCTTAGA ACTCAAAACT CTGGTTGCTC TTTAAAACTG 1246GCTCAAGAGA ATAAACTCAA ACTTGGGTGT TCATCATTGA AATTCCTGAC CCCACCACGT 1306CCCCAACCGT CCAGACTCTA CAGTGAGAGT GACACATATG ATGCTAGTAG ACTGCAGGCA 1366GTATCTGTTA TACACTGTAT AGACTGCAGA TTCGATCATG GGAGTGCTGC AATATAGAAA 1426TGTGACCTAT GTCTTTTTTA CTAGAGAATA TAGTGTGTAT ATAATTCCTA CATGAATTAT 1486GGTAACTGGG AACAGCATTG TAATTAAAAG ATTTGCAAAT GCTACTACAA GACAAAGACC 1546AGGTCATCCC TTTGTGAACT TGGTCGTAAA CATTTTTAGA ATCTCTATGA AGTCCAAGAA 1606AAACAAGATA ACTAAAATGA CATAATACTA AAGGGTGGAA AACAAGGAGC AATCGTATTT 1666TGTTATTAAG TTTTTAAGTA TCTTCAAAAG AACTTTTCCA GGGCTAGGGA GAAGGCTGAC 1726AGTCAAGAGG CTACTGAGTT TTTTTCCAGA GTTCTGAGTT CAATTCCCAG CAACTACATG 1786GTAGTCACAA CCATCTGTAA TGGACCCGAT GCCCTCTTCT GGTGTGTCTG AAGACAGCTA 1846CAGTGTACTC ACATACATAA AATAAGTAAA TCTTAAAAAA AAAAAAAAAA A 1897

Table 2 shows amino acid sequence homology of the BOG pRb binding domain(SEQ ID NO: 2) and casein kinase II consensus sequence to the Rb bindingproteins Human Papilloma Virus E7 (SEQ ID NO: 3), Simian Virus 40 largeT antigen (SEQ ID NO: 4), Adenovirus E1A (SEQ ID NO: 5) and RBP-1 (SEQID NO: 6) is illustrated by alignment of the Rb binding domain (LXCXE)(bold) and casein kinase II phosphorylation sites (underlined). BOG L PF M E T E - L H C D E K T I   (−44aa) - S T D D HPV16E7 D L Q P E T T DL Y C Y E Q - L N D       S S E E E SV40 LT N A F N E - E N L F C S EE - M P -       S S D D E AD5 E1A N L V P E V I D L T C H E A G F P P --     S D D E RBP-1 L I G P E T - - L V C H E V D L   (−8aa)  T S E I D

Table 3 shows the total amino acid sequence homology of rBOG (SEQ ID NO:2) and E7 (SEQ ID NO: 3) showing that rBOG is 38% homologous to E7. rBOGMVSPTKAVIVPGNGGGDVATHGWYGWVRKGLEQIPGFQCLAKNMPDPITA  50 HPV-E7MH-------------GDTPT-------------------LHEYMLD---- rBOGRESIWLPFMETELHCDEKTIIIGHSSGAIAAMRYAETHQVYALILVSAYT 100 HPV-E7-----LQPETTDLYCYEQ---LNDSSEE---------------------- rBOGSDLGDENERASGYFSRPWQWEKIKANCPHIIQFGSTDDPFLPWKEQQEVA 150 HPV-E7---EDEIDGPAG------QAEPDRAHY-NIVTFCCKCDSTLRLCVQSTHV rBOGDSWTPNCTNSLTVVTFRTQSSMN 173 Identity: 29.6% HPV-E7DIRTLEDLLMGTLGIVCPICSQKP  98 Similarity:  7.1%

Table 4 shows the cDNA coding sequence of human BOG. (SEQ ID NO: 7)ATGGTGTCCC CCAGCAAGGC AGTGATTGTT CCCGGGAAGA TAGGTGGGGA TGAGACCACC  60CACGGCTGGT ATGGCTGGGT GAAAAAGGAG CTGGAGAAGA TACCTGGTTT CCAGTGTTTG 120GCTAAAAACA TGCCCGACCC AATTACCGCG CGAGAGAGCA TCTGGCTGCC CTTCATGGAG 180ACAGAACTGC ACTGTGATGA GAAGACTATC ATCATTGGCC ACAGTTCCGG GGCCATCGCG 240GCCATGAGGT ATGCAGAAAC ACATCGAGTA TATGCTCTCA TATTGGTGTC TGCATACACA 300TCAGAGTTTG GAGATGAAAA TGAGCGTGCA AGTGGGTACT TCAGCCGCCC CTGGCAGTGG 360GAGAAGATCA AGGCCAACTG CCCTCACATT GTACAGTTTG GCTCTACTGA TGACCCCTTC 420CTTCCCTGGA AGGAACAACA AGAAGTGGCA GATAGCTGGA CGCCAAATTG TACAAATTCA 480CTGACCGTGG TCACTTTCAG AACACAGAGT TCCATGAACT GA 522

Table 5 shows the amino acid sequence of human BOG protein. (SEQ ID NO:8) MVSPSKAVIVPGKIGGDETTHGWYGWVKKELEKIPGFQCLAKNMP 050 DPITARESIWLPFMETELHCDEKTIIIGHSSGAIAAMRYAETHRVYALIL 100 VSAYTSEFGDENERASGYFSRPWQWEKIKANCPHIVQFGSTDDPFLPWKE 150 QQEVADSWTPNCTNSLTVVTFRTQSSMN 173

Table 6 shows the cDNA coding sequence of murine BOG. (SEQ ID NO: 9)ATGGCGTCCC CCAACAAGGC AGTGATTGTT CCTGGGAACG GAGGCGGGGA TGTGGCCACC 060CACGGCTGGT ATGGCTGGGT GAAAAAGGGG CTGGAGCAGA TTCCTGGTTT CCAGTGTTTG 120GCTAAAAACA TGCCTGACCC AATTACCGCG CGAGAGAGCA TCTGGCTGCC CTTCATGGAG 180ACAGAGCTGC ACTGTGACGA GAAGACCATC ATCATAGGCC ACAGTTCCGG GGCCATCGCA 240GCCATGAGGT ATGCAGAGAC ACATCAGGTA TACGCTCTCG TATTGGTGTC TGCATACACA 300TCAGACTTGG GAGATGAAAA TGAGCGTGCA AGTGGGTACT TCAGCCGCCC CTGGCAGTGG 360GAGAAGATCA AGGCCAACTG CCCTCACATT ATACAGTTTG GCTCTACTGA TGACCCCTTC 420CTTCCCTGGA AGGAACAACA AGAAGTGGCA GATAGCTGGA CGCCAAATTG TACAAATTCA 480CTGACCGTGG TCACTTTCAG AACACAGAGT TCCATGAACT GA 522

Table 7 shows the amino acid sequence of murine BOG protein. (SEQ ID NO:10) MASPNKAVIVPGNGGGDVATHGWYGWVKKGLEQIPGFQCLAKNMP 050 DPITARESIWLPFMETELHCDEKTIIIGHSSGAIAAMRYAETHQVYALVL 100 VSAYTSDLGDENERASGYFSRPWQWEKIKANCPHIIQFGSTDDPFLPWKE 150 QQEVADSWTPNCTNSLTVVTFRTQSSMN 173

Table 8 shows a comparison of the amino acid sequence of the murine (SEQID NO: 10), human (SEQ ID NO: 8) and rat BOG (SEQ ID NO: 2) proteins.Murine MASPNKAVIVPGNGGGDVATHGWYGWVKKGLEQIPGFQCLAKNMPDPITA 050 RatMVSPTKAVIVPGNGGGDVATHGWYGWVRKGLEQIPGFQCLAKNMPDPITA HumanMVSPSKAVIVPGKIGGDETTHGWYGWVKKELEKIPGFQCLAKNMPDPITA MurineRESIWLPEMETELHCDEKTIIIGHSSGAIAAMRYAETHQVYALVLVSAYT 100 RatRESIWLPEMETELHCDEKTIIIGHSSGAIAAMRYAETHQVYALILVSAYT HumanRESIWLPFMETELHCDEKTIIIGHSSGAIAAMRYAETHRVYALILVSAYT MurineSDLGDENERASGYFSRPWQWEKIKANCPHIIQFGSTDDPFLPWKEQQEVA 150 RatSDLGDENERASGYFSRPWQWEKIKANCPHIIQFGSTDDPFLPWKEQQEVA HumanSEFGDENERASGYFSRPWQWEKIKANCPHIVQFGSTDDPFLPWKEQQEVA MurineDSWTPNCTNSLTVVTFRTQSSMN 173 Rat DSWTPNCTNSLTVVTFRTQSSMN HumanDSWTPNCTNSLTVVTFRTQSSMN

1. An isolated polypeptide comprising BOG polypeptide comprising (i) atleast 90% amino acid sequence identity with SEQ ID NO: 8; (ii) aretinoblastoma gene product (pRB) binding motif; and (iii) at least onecasein kinase II phosphorylation motif; wherein the polypeptide bindspRB and displaces the transcriptional factor E2F-1 bound to pRB.
 2. TheBOG polypeptide claim 1, wherein said casein kinase II phosphorylationmotif is located downstream of the pRb binding motif.
 3. The BOGpolypeptide of claim 2, further comprising a second casein kinase IIphosphorylation motif, said second casein kinase II phosphorylationmotif being located upstream of the pRb binding motif.
 4. The BOGpolypeptide of claim 1 joined to a detectable label.
 5. The BOGpolypeptide of claim 4, wherein the detectable label includes aradioactive isotope, an enzyme, a chromophore or a mixture thereof.
 6. Achimeric molecule comprising the BOG polypeptide of claim 1 fused to aheterologous amino acid sequence.
 7. An isolated polypeptide comprising:i) at least 95% amino acid sequence identity with SEQ ID NO:8 ii) aretinoblastoma gene product (pRB) binding motif; and iii) at least onecasein kinase II phosphorylation motif; wherein the polypeptide bindspRB and displaces the transcriptional factor E2F-1 bound to pRB.
 8. Thepolypeptide of claim 7 comprising the amino acid sequence of SEQ IDNO:8.
 9. The polypeptide of claim 7, wherein said casein kinase IIphosphorylation motif is located downstream of the pRB binding motif.10. The polypeptide of claim 9 further comprising a second casein IIphosphorylation motif, said second casein kinase II phosphorylationmotif being located upstream of the pRB binding motif.
 11. Thepolypeptide of claim 7 joined to a detectable label.
 12. The polypeptideof claim 11, wherein the detectable label comprises a radioactiveisotope, an enzyme, a chromophore or a mixture thereof.
 13. Thepolypeptide of claim 7 further comprising a heterologous amino acidsequence.
 14. The polypeptide of claim 13, wherein the heterologousamino acid is a tag polypeptide.
 15. The polypeptide of claim 13,wherein the heterologous amino acid sequence is that of animmunoglobulin constant region.
 16. The polypeptide of claim 13, whereinthe heterologous amino acid sequence is maltose binding protein.
 17. Anisolated polypeptide comprising a polypeptide having 95% sequenceidentity to the amino acid sequence of SEQ ID NO:8, wherein the isolatedpolypeptide binds the retinoblastoma gene product (pRB) and displacesthe transcriptional factor E2F-1 bound to pRB.
 18. The isolatedpolypeptide of claim 17, comprising the amino acid sequence of SEQ IDNO:8.
 19. The isolated polypeptide of claim 1, comprising a polypeptidehaving 95% sequence identity to the amino acid sequence of SEQ ID NO:8.20. The isolated polypeptide of claim 19, comprising the amino acidsequence of SEQ ID NO:8.